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G Tsujimoto

Publications and source records attributed to G Tsujimoto.

At least 37 records · Page 2Linked to original sources
PubMed2001

[A model for clinical study based on genome science--trials in disorders of the immune system and allergies].

Disorders of the immune system, such as allergies, have multi-factorial etiologies that include both genetic and environmental components. The recent advances in genome science have facilitated two strategies for studying the genetic basis of disease: (1) systematic analysis of gene expression profiles and (2) comprehensive analysis of gene variations, such as polymorphisms. Here, we describe a unique research institute, Genox Research Inc., that can relate the clinical profile of a patient to genotyping and molecular profiling. Systematic gene expression analyses using differential display have been performed to explore genes related to allergy, and revealed 93 differentially expressed candidate genes in T-cells. Also, a single nucleotide polymorphism(SNP) analysis project has been designed to mine disease-related and/or drug-response-related genes involved in allergic disorders using biochip technologies. As exemplified above, clinical studies based on these applications of genome science would be of considerable value in clarifying our understanding of multi-gene disorders.

Gene Expression Profiling↗
PubMed2001

['Millennium Project' of MHLW].

Human Genome Project (information and technology) provides insights so profound that it has the ability to change the way we understand, predict, prevent, diagnose, and treat disease. Because SNPs, single nucleotide polymorphisms, are the most common type of polymorphism, they can have significant effects on both susceptibility to disease as well as drug response. In Japan, 'Millennium Project' based on SNPs has started in 2000, and National Research Centers of MHLW are performing genome-wide association study on five common diseases (dementia, cancer, diabetes mellitus, hypertension, and asthma/allergy). National Children's Hospital group studies asthma/allergy and its drug therapy. Here the overview of 'Millennium Project' and the actual research framework of National Children's Hospital group is reviewed.

Asthma↗
PubMed2000

Cloning and characterization of the promoter of murine cytohesin-1 gene.

Mouse cytohesin-1 (CTH-1) is a guanine nucleotide exchange factor, specific for ADP ribosylation factors. The CTH-1 gene promoter was characterized by deletion mapping and mutational analysis. The region between -101 and -38 relative to the transcription start site showed the essential promoter activity when transfected into both NIH3T3 and COS7 cells. The nucleotides of the core promoter region contain three tandem GC boxes, which can offer potential binding sites for the ubiquitous transcription factor Sp-1 family. Mutational analysis revealed that these tandem GC boxes are indispensable for activation of the gene transcription.

3T3 Cells↗
PubMed2000

Identification of three human type-II classic cadherins and frequent heterophilic interactions between different subclasses of type-II classic cadherins.

We identified three novel human type-II classic cadherins, cadherin-7, -9 and -10, by cDNA cloning and sequencing, and confirmed that they interact with catenins and function in cell-cell adhesion as do other classic cadherins. Cell-cell binding activities of the eight human type-II classic cadherins, including the three new molecules, were evaluated by long-term cell-aggregation experiments using mouse L fibroblast clones transfected with the individual cadherins. The experiments indicated that all the type-II cadherins appeared to possess similar binding strength, which was virtually equivalent to that of E-cadherin. We next examined the binding specificities of the type-II cadherins using the mixed cell-aggregation assay. Although all of the type-II cadherins exhibited binding specificities distinct from that of E-cadherin, heterophilic interactions ranging from incomplete to complete were frequently observed among them. The combinations of cadherin-6 and -9, cadherin-7 and -14, cadherin-8 and -11, and cadherin-9 and -10 interacted in a complete manner, and in particular cadherin-7 and -14, and cadherin-8 and -11 showed an indistinguishable binding specificity against other cadherin subclasses, at least in this assay system. Although these data were obtained from an in vitro study, they should be useful for understanding cadherin-mediated mechanisms of development, morphogenesis and cell-cell interactions in vivo.

Amino Acid Sequence↗
PubMed2000

Pharmacological characterization of a novel AVP(4-9) binding site in rat hippocampus.

pGlu-Asn-Cys (Cys)-Pro-Arg-Gly-NH(2) (AVP(4-9)), a major metabolite C-terminal fragment of Arginine(8)-vasopressin (AVP), improves the disruption of the learning and memory, and is a far more potent in the mnemonic function than AVP. In this study, we pharmacologically characterized its putative binding site and mechanism of intracellular signaling. Radioligand binding assay showed that [35S]AVP(4-9) could detect specific binding sites in the rat hippocampus membrane preparations, and the binding site was specifically displaced by AVP(4-9) but not by either V(1) or V(2) antagonists. Furthermore, [35S]AVP(4-9) could not detect the cloned rat V(1a), V(1b) and V(2) vasopressin receptors. Even at a low doses (10-100 pM), AVP(4-9) caused an increase in both inositol(1,4, 5)-trisphosphate (Ins(1,4,5)P(3)) and intracellular calcium concentrations ([Ca(2+)](i)) in rat hippocampal cells. The AVP(4-9)-induced [Ca(2+)](i) increase was partially inhibited by the absence of Ca(2+) or by Ca(2+)-channel blocker, suggesting that AVP(4-9) caused the [Ca(2+)](i) increase via release from intracellular calcium store as well as influx from extracellular calcium. For the first time, this study provides evidence to show that AVP(4-9) activates Ins(1,4,5)P(3)/[Ca(2+)](i) pathway through a novel type of receptor in rat hippocampus, which might be potentially important in improving the mnemonic function.

Animals↗
PubMed2000

Key amino acids of vasopressin V1a receptor responsible for the species difference in the affinity of OPC-21268.

A non-peptide, vasopressin V1a receptor-selective antagonist, OPC-21268, exhibited a markedly higher affinity for the rat V1a receptor (Ki = 380 nM) than for the human V1a receptor (Ki = 140 microM). To delineate the region responsible for the high affinity binding of OPC-21268 for the rat V1a receptor, we have constructed a series of chimeric human and rat V1a receptors, and examined the chimeric and point-mutated receptors by competitive radioligand binding analysis. The results showed that the transmembrane domain (TMD) VI-VII of the vasopressin V1a receptor, in particular the amino acid residue Ala-342 in TMD VII, is the major component conferring the rat-selective binding of OPC-21268 to the V1a receptor.

Amino Acid Sequence↗
PubMed2000

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes.

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.

Animals↗
PubMed2000

Evaluation of heterologous insulator function with regard to chromosomal position effect in the mouse blastocyst and fetus.

Insulators are located at the boundaries of differentially regulated genes and delimit their interactions by establishing independent chromatin structures. Recently, an insulator sequence has been found in the 5'-flanking region of arylsulfatase (ARS) gene from sea urchin. To investigate functional conservation of this ARS insulator in mice, we performed blastocyst assays to evaluate the effect of this insulator on the chromosomal position effect, quantitatively. We constructed transgenes that have a luciferase gene under the control of the CMV-IE enhancer and the human elongation factor 1 alpha promoter in the presence or absence of the ARS insulator in both flanking regions. These transgenes were microinjected into 1-cell mouse embryos and luciferase activity was measured at the blastocyst stage. We found that the presence of ARS insulator sequence doubled the number of luciferase-expressing blastocysts, and that the proportion of the blastocysts with high-level expression (> or = 1 x 10(4) relative light units (RLU)) was increased more than tenfold. In the case of transgenic fetuses, however, the presence of ARS insulator did not seem to improve transgene expression. These results suggest that the sea urchin ARS insulator confers position-independent expression driven by the human elongation factor 1 alpha promoter, at least in the blastocyst stage of the mouse.

Animals↗
PubMed2000

Impaired coronary microvascular function in diabetics.

Global and regional myocardial uptake was determined with technetium-99m tetrofosmin and a 4 hour exercise (370 MBq i.v.) and rest (740 MBq i.v.) protocol, in 24 patients with non-insulin dependent diabetes mellitus and in 22 control subjects. The purpose of this study was to evaluate impaired coronary microvascular function in diabetics by measurement of % uptake increase in myocardial counts. The parameter of % uptake increase (deltaMTU) was calculated as the ratio of exercise counts to rest myocardial counts with correction of myocardial uptake for dose administered and physical decay between the exercise study and the rest study. Global deltaMTU was significantly lower in the diabetics than in control subjects (14.4 +/- 5.4% vs. 21.7 +/- 8.5%, p < 0.01). Regional deltaMTU in each of 4 left ventricular regions (anterior, septal, inferior, posterolateral) was significantly lower in the diabetic group than in the control group (p < 0.01) respectively, but there were no significant differences between deltaMTU in the 4 left ventricular regions in the same group. deltaMTU was useful as a non-invasive means of evaluating impaired coronary microvascular function in diabetics.

Blood Pressure↗
PubMed2000

Distribution of alpha1-adrenoceptor subtype mRNA and identification of subtype responsible for renovascular contraction in human renal artery.

This study was intended to quantify the amounts of the alpha1-adrenoceptor subtype mRNAs in human renal artery and to demonstrate the distribution of receptor subtypes responsible for the contraction of the renal artery. RNase protection assay showed that the mean amount of alpha1a mRNA was much greater than that of alpha1b or alpha1d mRNAs in both the main and branch renal arteries. However, the abundance of alpha1a mRNA in human renal artery was much less than in our previous data in the prostate. In situ hybridization showed that all alpha1 subtype mRNAs were localized in the smooth muscle cells of the tunica media of the artery, and the distribution pattern of these three mRNAs in the main artery was the same as in the branch artery. However, the intensity of signals for alpha1d and alpha1b antisense RNAs probes was lower than that for the alpha1a antisense RNA probe. In the functional study, concentration-response curves to noradrenaline pretreated with KMD-3213, an alpha1A/L-adrenoceptor selective antagonist, seemed to be biphasic in nature. Chloroethyclonidine (CEC) failed to inactivate the noradrenaline-induced contraction, and prazosin showed relatively low affinity with a pA2 value of 8.8. These data suggest that the alpha1A/L-adrenoceptor mediates primarily those responses to noradrenaline in this artery. The other alpha1-adrenoceptor subtypes could also mediate the secondary contractile response to noradrenaline in this artery.

Adrenergic alpha-Antagonists↗
PubMed2000

Both alpha(1A)- and alpha(1B)-adrenergic receptor subtypes couple to the transient outward current (I(To)) in rat ventricular myocytes.

1. Regulation of transient outward current (I(To)) by alpha(1)-adrenergic (alpha(1)AR) plays a key role in cardiac repolarization. alpha(1)ARs comprise a heterogeneous family; two natively expressed subtypes (alpha(1A) and alpha(1B)) and three cloned subtypes (alpha(1a), alpha(1b) and alpha(1d)) can be distinguished. We have examined the electrophysiological role of each alpha(1)AR subtype in regulating I(To) in isolated rat ventricular myocytes. 2. Reverse transcription-PCR study revealed the presence of three subtype mRNAs (alpha(1a), alpha(1b) and alpha(1d)) in rat myocytes. 3. Radioligand binding assay using [(125)I]-HEAT showed that the inhibition curves for alpha(1A)AR-selective antagonists (WB4101, 5-methylurapidil, (+)-niguldipine and KMD-3213) in rat ventricles best fit a two-site model, with 30% high and 70% low affinity binding sites. The high affinity sites were resistant to 100 microM chloroethylclonidine (CEC), while the low affinity sites were highly inactivated by CEC. 4. Whole cell voltage clamp study revealed that methoxamine reduced a 4-aminopyridine(4-AP)-sensitive component of I(To) in the isolated rat ventricle myocytes. Lower concentrations of KMD-3213 (1 nM) or 5-MU (10 nM) did not affect the methoxamine-induced reduction of I(To). On the other hand, CEC treatment (100 microM) of isolated myocytes reduced the methoxamine-induced reduction of I(To) by 46%, and the remaining response was abolished by lower concentrations of KMD-3213 or 5-MU. 5. The results indicate that rat ventricular myocytes express transcripts of the three alpha(1)AR subtypes (alpha(1a), alpha(1b) and alpha(1d)); however, two pharmacologically distinct alpha(1)AR subtypes (alpha(1A) and alpha(1B)) are predominating in receptor populations, with approximately 30% alpha(1A)AR and 70% alpha(1B)AR. Although both alpha(1A) and alpha(1B)AR subtypes are coupled to the cardiac I(To), alpha(1B)ARs predominantly mediate alpha(1)AR-induced effect.

Adrenergic alpha-Agonists↗
PubMed2000

Characterization of a novel nonpeptide vasopressin V(2)-agonist, OPC-51803, in cells transfected human vasopressin receptor subtypes.

We discovered the first nonpeptide arginine-vasopressin (AVP) V(2)-receptor agonist, OPC-51803. Pharmacological properties of OPC-51803 were elucidated using HeLa cells expressing human AVP receptor subtypes (V(2), V(1a) and V(1b)) and compared with those of 1-desamino-8-D-arginine vasopressin (dDAVP), a peptide V(2)-receptor agonist. OPC-51803 and dDAVP displaced [(3)H]-AVP binding to human V(2)- and V(1a)-receptors with K(i) values of 91.9+/-10.8 nM (n = 6) and 3.12+/-0.38 nM (n = 6) for V(2)-receptors, and 819+/-39 nM (n = 6) and 41.5+/-9.9 nM (n = 6) for V(1a)-receptors, indicating that OPC-51803 was about nine times more selective for V(2)-receptors, similar to the selectivity of dDAVP. OPC-51803 scarcely displaced [(3)H]-AVP binding to human V(1b)-receptors even at 10(-4) M, while dDAVP showed potent affinity to human V(1b)-receptors with the K(i) value of 13.7+/-3.2 nM (n = 4). OPC-51803 concentration-dependently increased cyclic adenosine 3', 5'-monophosphate (cyclic AMP) production in HeLa cells expressing human V(2)-receptors with an EC(50) value of 189+/-14 nM (n = 6). The concentration-response curve for cyclic AMP production induced by OPC-51803 was shifted to the right in the presence of a V(2)-antagonist, OPC-31260. At 10(-5) M, OPC-51803 did not increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells expressing human V(1a)-receptors. On the other hand, dDAVP increased [Ca(2+)](i) in HeLa cells expressing human V(1a)- and V(1b)-receptors in a concentration-dependent fashion. From these results, OPC-51803 has been confirmed to be the first nonpeptide agonist for human AVP V(2)-receptors without agonistic activities for V(1a)- and V(1b)-receptors. OPC-51803 may be useful for the treatment of AVP-deficient pathophysiological states and as a tool for AVP researches.

Arginine Vasopressin↗
PubMed2000

Effects of castration on contraction and alpha(1)-adrenoceptor expression in rat prostate.

1. The prostate function is regulated by androgens and alpha-adrenergic activity. Clinically, antiandrogens and/or alpha(1)-adrenergic antagonists are commonly used to treat symptomatic prostatic hypertrophy. To elucidate the effects of androgen deprivation on prostate contractility via alpha(1)-adrenoceptor, the characteristics and expression of alpha(1)-adrenoceptors were examined in castrated rats. 2. Isolated prostate strips from intact and castrated rats were subjected to a phenylephrine stimulated contraction. Prazosin (10 nM), [(3)H]-prazosin and phenoxybenzamine (3 - 300 nM) were used for inhibition assay, receptor characterization and partial alkylation of alpha-adrenoceptor, respectively. The mRNA content of three subtypes of alpha-adrenoceptors was determined by reverse transcription combined with polymerase chain reaction (RT - PCR). 3. Contractile response to phenylephrine increased in castrated rats, which could be explained by a relative increase of the stromal component. A lowered contraction potency was also noted in castrated rats. Receptor binding assay indicated minimal changes in the affinity or density of alpha(1)-adrenoceptor. Escalating alkylation of the alpha(1)-adrenoceptor population resulted in a rightward shift in the contraction-response curves before depressing maximal contractile force, and the suppression was detected at lower doses in castrated rats. RT - PCR study confirmed the expression of three types of alpha(1)-adrenoceptor, alpha(1a), alpha(1b) and alpha(1d)-adrenoceptors, in intact rat prostate, and revealed that alpha(1a)-adrenoceptor, but not alpha(1b) or alpha(1d)-adrenoceptors, was down-regulated in castrates. 4. The results show that androgen deprivation suppressed alpha(1)-adrenergic contractility of rat prostate strips, and the suppression was associated with down-regulation of receptor reserve for the alpha(1a)-adreneroceptor population expressed in intact rat prostate.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2000

Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells.

ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R >> P2X(2a)R >> P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.

Animals↗
PubMed2000

Distribution of geriatric disease-related genotypes in the National Institute for Longevity Sciences, Longitudinal Study of Aging (NILS-LSA).

Phenotypes of various genes related to geriatric diseases and the aging process were assessed in the National Institute for Longevity Sciences, Longitudinal Study of Aging (NILS-LSA). The subjects were 1,297 participants in the NILS-LSA. They were community-living males and females aged 40 to 79 years who were randomly selected from the area of the NILS. Genotypic and allelic frequencies of genes in the subjects were analyzed. Age and gender differences in the distribution of genotypes were also tested. The genotypic frequencies were as follows: (1) Angiotensin I-converting enzyme (ACE) genotype was I/I 46.2%, I/D 38.3% and D/D 15.5%. (2) alpha 1-adrenoreceptor genotype was C/C 84.4%, C/T 12.7%, and T/T 3.0%. (3) Apolipoprotein E genotype was epsilon 2/epsilon 2 0%, epsilon 2/epsilon 3 7.9%, epsilon 3/epsilon 3 70.0%, epsilon 3/epsilon 4 20.8%, epsilon 2/epsilon 4 0%, and epsilon 4/epsilon 4 1.4%. (4) Cholecystokinin type-A receptor (CCKAR) nucleotide-81 (nt-81) genotype was A/A 59.1%, A/G 35.1%, and G/G 5.9%. The CCKAR nucleotide-128 genotype (nt-128) was G/G 74.3%, G/T 23.6%, and T/T 2.2%. The combination of nucleotide (nt-81, nt-128) was (A/A, G/G) 59.1%, (A/G, G/G) 14.1%, (G/G, G/G) 1.1%, (A/G, G/T) 21.0%, (G/G, G/T) 2.6%, and (G/G, T/T) 2.1%. There were no subjects with (A/A, G/T), (A/A, T/T) or (A/G, T/T) genotypic combinations. (5) beta 3-adrenoreceptor genotype was T/T 66.8%, T/A 28.5%, and A/A 4.7%. (6) Dihydrolipoamide succinyltransferase (DLST) nucleotide 19117 genotype was A/A 25.1%, A/G 49.7%, and G/G 25.1%. The DLST nucleotide 19183 genotype was C/C 55.8%, C/T 38.2%, and T/T 5.9%. The combination of nucleotide (nt19117, nt19183) was (A/A, C/C) 6.7%, (A/G, C/C) 24.1%, (G/G, C/C) 25.1%, (A/G, C/T) 25.6%, (A/A, T/T) 5.9%, and (A/A, C/T) 12.6%. There were no subjects with (A/G, T/T), (G/G, T/T) or (G/G, T/C) genotypic combinations. (7) Transforming growth factor-beta 1 genotype T/T 35.2%, T/C 44.6%, and C/C 20.2%. (8) The platelet-activating factor acetylhydrolase genotype was M/M 71.7%, M/m 27.2%, and m/m 1.2%. The mitochondria DNA 5178 genotype A was 42.1% and C was 57.9%. There were no significant gender or age differences in tested genotypic and allelic distribution except for the DLST and apolipoprotein E. Differences in the genotypic frequencies of distribution using the Hardy-Weinberg equilibrium were significant in the ACE and alpha 1-adrenoreceptor genotypes.

Adult↗
PubMed2000

[Non-invasive assessment of coronary microvascular function in patients with syndrome X using exercise and rest myocardial SPECT with 99mTc-tetrofosmin].

The purpose of this study was to evaluate impaired coronary microvascular function in Syndrome X (Sx) by measuring % uptake increase in myocardial counts. Global and regional myocardial uptake was determined with 99mTc-tetrofosmin and a 4-hour exercise (370 MBq i.v.) and rest (740 MBq i.v.) protocol, in 11 patients with anginal syndrome and normal coronary arteriograms (Sx) and in 10 control subjects (C). The parameter of % uptake increase (delta MTU) was calculated as the ratio of exercise counts to rest myocardial counts with correction of myocardial uptake for dose administered and physical decay between the exercise study and the rest study. Global delta MTU was significantly lower in Sx than C (12.9 +/- 5.4% vs. 22.3 +/- 10.8%, p < 0.05). Regional delta MTU in each of 4 left ventricular regions (anterior, septal, inferior, posterolateral) was significantly lower in Sx than in C (p < 0.05), except for the inferior region. However, there were no significant differences between delta MTU in the 4 left ventricular regions in the same group. None of the patients with Sx exhibited an ischemic pattern in the ST-segment/heart rate loop. delta MTU was useful as a non-invasive means of evaluating impaired coronary microvascular function in Sx.

Aged↗